The techniques developed under the category of recombinant DNA (RDNA) technology have proven to be very powerful for molecular investigations (e.g., study of the molecular bases or mechanisms of cellular function). They are most often used for qualitative analysis.
Many important genetic conditions involve quantitative changes in the amount of DNA present in cells; these are not accompanied by related qualitative alterations (such as changes in the type of cellular proteins). Presently-available RDNA methods, however, are not useful for assessing such quantitative changes in cells on an individual basis for diagnosis or exploration of genetic conditions whose causality is unknown.
In standard RDNA methods, DNA molecules are enzymatically digested to produce small fragments; the resulting fragments are separated by size on a gel, through application of an electric field. They are subsequently transferred to a support through a procedure called blotting. Specific fragments of interest can be detected through use of a defined nucleic acid segment which has a sequence homologous to that of the fragment of interest and which has been labelled in a manner which makes it identifiable. Such segments, when inserted into a vector, are referred to as probes and are often labelled radioactively (e.g., by substitution of some of their phosphorous atoms by radioactive isotopes). When the labelled probes are combined under appropriate conditions with sample DNA (i.e., DNA to be analyzed) they will complex or hybridize with a DNA fragment which has a homologous sequence, resulting in formation of a "hybrid" molecule which is radioactive (i.e., radioactively labelled). Localization of the hybrid is detected by allowing the decay product from the radioactivity to interact with a closely-applied piece of X-ray film. When the film is developed, the silver grains that have been deposited by the beta decay energization produce a dark image on the film. The same approach can be used for RNA. Maniatis, T. et al., Molecular Cloning: A Laboratory Approach, Cold Spring Harbor Laboratory Press (1982).
The amount of radioactivity emitted at the point of attachment of a specific probe can be measured by determination of the density of the image created on the film in a given period of time. This then permits a measure of the amount of substance to which the radioactively labelled probe was attached. This technique can be used in principle to measure the quantity of a nucleic acid of interest in a given sample and has been employed for estimates of relative amounts of DNA and RNA in many systems. However, such techniques have methodological limitations which mean that they are not useful in investigations or diagnostic assessments which require accurate measurement of quantitative changes for individual samples.
Current models of genetic disorders, such as that for sickle cell anemia, include mutational events at the DNA level, which result in the transcription of an altered RNA and ultimate translation into an abnormal protein which, in turn, results in a disease state. However, there are many, varied genetic abnormalities which involve only quantitative changes in DNA present in cells and no identifiable mutational event, altered RNA, or abnormal protein production. Some of these are classed as chromosomal abnormalities; examples of these are Down Syndrome and several sex chromosome inbalances, such as Klinefelter's Syndrome and Turner's Syndrome.
Down Syndrome has been shown to involve an increase in relative amounts of a specific segment of chromosome 21. Down Syndrome occurs most commonly in the form of Trisomy 21. Normal cells have 2 complete chromosome 21's per cell nucleus; in Trisomy 21, cells have 3 complete chromosome 21's. The full effect of Down Syndrome has been shown to require triplication of only a limited portion of chromosome 21, which has been localized to chromosome band 21q22. When Trisomy 21 occurs, it can be detected microscopically, thus making diagnosis possible by karyotypic analysis. When Down Syndrome is caused by replication of a limited portion of chromosome 21, it is often difficult to detect karyotypically, even with intense and specialized staining examinations. Prenatal samples to be analyzed using presently-available methods are obtained through amniocentesis, a procedure which has a number of drawbacks. For example, the procedure cannot generally be carried out until the sixteenth week of pregnancy; cell culture and analysis of the fluid obtained requires three to four weeks; a 10% failure rate occurs, which requires drawing a new sample and an additional 3-4 weeks to culture the cells. In addition, there is a certain rate of incorrect diagnosis (5 per 1000 cases). Milunsky, A., Prenatal Diagnosis of Hereditary Disorders (2nd ed.) Charles Thomas Press, Springfield, Ill., (1979); Hirschhorn, K., In: Methods in Cell Biology, Latt, S. and G. Darlington (ed.), 26: 1-9 (1982).
There have been a number of attempts previously at adapting RDNA methods for the diagnosis of Down Syndrome, but each has not succeeded. The difficulties involved are exemplified in the following results of one such effort. Devine, E. et al., Annals of the New York Academy of Sciences, 450: 69-83 (1985).
TABLE 1 ______________________________________ Quantitation of Hybridization to Normal and Trisomy 21 DNA Ratio of .beta.-Globin 21 21 DNA/ DNA Fragment .beta.-Globin DNA ______________________________________ Trisomy 21 633 364 .575 558 132 .240 546 102 .190 528 180 .340 790 520 .660 2.005 Normal 438 130 .300 975 238 .240 460 143 .310 507 108 .200 986 238 .240 1.290 ______________________________________ Combined Trisomy/Normal = 1.55
In this case, the important methodological problem was the lack of ability to control the exposure of the film to the radioactive signal. As a result, individual measurements varied widely. As shown in Table 1, overlap occurs in individual results, between normal and Down Syndrome individuals, despite the fact it was known that the DNA amounts differed because of the chromosomal analyses. To overcome this variability, multiple sample results were pooled. The same approach has been used by others in attempts to quantify DNA amounts in Down Syndrome.
RDNA quantification was reported for Cat Eye syndrome. McDermid H., et al., Science, 232: 646-648, (1986). However, multiple tests (9.times.) were done for each determination of the ratio of test chromosome to reference chromosome. The pooled mean ratio from the normal individuals was then normalized for comparison with pooled repeats of the affected people. However, pooled results cannot form the basis of a diagnostic test, which must be reliable at an individual level. Pooling of results assumes that the variability of results found upon multiple measurements will approximate a true mean value (i.e., that repeated data are subject to random variability). There is no evidence in support of this.
The variability in multiple measurements reported occurs because of the lack of control of the signal deposited on the film. Presently, however, there is no method to determine whether the image was over- or underexposed. Either produces uncontrolled results which are not expected to vary randomly. It is generally assumed that RDNA methods cannot be used for diagnosis of Down Syndrome or other conditions whose biochemical basis is a quantity of DNA present in cells of affected individuals.
In the method illustrated in simplified form in FIG. 1, DNA 22 is isolated from a tissue sample and purified. As represented in FIG. 1A, in which chromosome 21 is shown, DNA is made of pairs of strands 24, 26, which form a helical configuration; each strand of DNA is made of nucleotides 28, whose ordering in each molecule of DNA forms a specific sequence. Restriction enzymes act on specific small sequences in DNA and can be used to cut DNA, as shown in FIG. 1B. If properly-chosen restriction enzymes are used, the DNA of chromosome 21 will be cut so as to produce a number of DNA fragments originating from band 21q22.
The DNA fragments resulting from the action of (i.e., digestion by) restriction enzymes are separated on the basis of size by immersion in a gel 30 and application of an electric field as represented in FIG. 1C. As shown in FIG. 1D, the molecules are transferred to a support 32, without modifying their relative locations on the gel. The DNA molecules, immobilized on the support, are treated to separate them into their constituent strands. Cloned recombinant plasmids 36 are prepared, using known techniques; such clones contain copies of DNA fragments identified as originating from band 21q22 inserted into a vector. The cloned DNA fragments and their vectors are radioactively labelled and can be used as labelled probes which will hybridize to the DNA 34 (shown attached to support 32 in FIG. 1E) whose separated strands have a nucleotide sequence complementary to the strands of the inserted probe sequence. As a result of hybridization of a strand of probe DNA 36 with the complementary nucleotide sequences on a strand of the genomic DNA on the support 32, the homologous sequences of interest become identified. This approach has serious inherent difficulties for quantitation, however, because of the exquisite sensitivity of detection (in the nanogram range) it makes possible. It is very difficult to compare the amount of label attached to two different samples because the variation introduced in loading the samples onto the gel will often be greater than the difference being evaluated.
As represented in FIG. 1F, in standard methods, an image 40 from a radioactively labelled DNA probe attached to a genomic DNA fragment on a blot is created by exposure of an X-ray film 38 to the support. A densitometric tracing is then made of the developed film. If the exposure can be controlled, the area subsumed under the peaks of the tracing can be directly proportional to the amount of radioactivity present in the probes hybridized to DNA fragments (attached to the support) which contain homologous sequences. The amount of radioactivity deposited on the film is a direct measure of the quantity of DNA present in the sample with controlled exposure.
A critical problem exists because of the inherent non-linearity of the film response relative to exposure (known as the H-D curve in classical optics). As illustrated by FIG. 2, the film registers a linear response only in a central range of exposures. Below or above that range of exposures, the images deposited on the film are not linearly related to the signal. In those cases, the quantity of material generating the radioactive signal cannot be directly measured. Complex computer calculations based on the densitometer readings have been attempted in an effort to assure that measurements are made in the linear range of the film response. However, those calculations have not been satisfactory.
In spite of the fact that recombinant DNA technology has been available for over ten years and the optimistic predictions of its applicability to diagnosis of genetic diseases, it has had no direct application for quantitatively-based conditions. This is due to the problem described above, the lack of a sufficiently sensitive and reliable method for quantification of changes in samples at the DNA molecular level. Such a quantitative approach would be very useful in diagnosing genetic disorders in which there is an alteration in quantity of DNA and could be valuable in exploring genetic conditions whose basis is not understood. For most genetic disorders, there is no specific genetic or biochemical characterization available.
Development of such an approach would be of particularly great value in a medical and public health context in that it would make it possible to diagnose, both prenatally and postnatally, conditions whose genetic basis lies in a quantitative change in the amount of DNA (or RNA) present in cells.